title: Trauma4

city: Shantou

first: Huixiong

last: Deng

address: Xinling 22 Rd

department: Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology

institute: Shantou University Medical College

data_processing:
The image data measured by the high-throughput sequencer are converted into sequence data (reads) by CASAVA base recognition. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing N base and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Assembly: mm10 Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 (v2.0.5) and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5). We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes RPKM values  for each Sample

source_name: Brian

growth_protocol: C57BL/6J mice were obtained from Beijing VitalLiver Laboratory Animal Technology Co., LTD. (Beijing, China). All experiments followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Standards for Animal Care and Use of Research at Shantou University Medical School (approval grant no SUMC2022-413). Pregnant C57BL/6J mice, aged between 12 and 18 weeks, were housed in a controlled environment with a temperature of 21 ± 2 ℃, humidity ranging from 30% to 70%, and a 12-hour light/dark cycle. They had ad libitum access to food and water. Within 24 hours of birth, neonatal C57BL/6J mice were anesthetized using isoflurane (R510-22, RWD Life Science, China) at an induction concentration of 5% and maintenance concentration of 2.5%. Brain tissue was extracted, and cortical neurons were isolated. The cerebral cortices, excluding the hippocampus and dura mater, were treated with 2 ml of papain (P3125, Sigma, USA) diluted to 60 U/ml at 37°C for 20 minutes to obtain cells. Subsequently, 1 × 106 cells were cultured on poly-D-lysine-treated glass coverslips (E607014-0002, Industrial Biotech, China) in 35 mm cell culture dishes/plates, supplemented with 2 ml of neurobasal medium in a 5% CO2 / 95% air atmosphere.100 GluamaxTM-1 (2003971, Gibco, USA) and SM1 (05711, stem cell, USA) were added to 2% of the neurobasal medium (05790, stem cell, USA) immediately before use. Half of the medium was changed every three days. On day 1, arabinosylcytosine (5 μmol, 147-94-4, MERCK, UK), a selective inhibitor of DNA synthesis, was added to the culture and incubated for 24 hours to minimize glial contamination. All experiments were conducted between day 8 and day 10.

extract_protocol:
RNA was harvested using Trizol(204202, ambion, USA), total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA,USA).
RNA sample preparations. Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA.Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. In order to ensure the quality of the library, the library needs to be tested. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is higher than that of 2nM) to ensure the quality of the library.

treatment_protocol: Cells were cultured in 24-well plates for 7 days. The medium was then replaced, and the cells were scratched and treated with TGF-β1 (10 ng/ml). After 24 hours, the supernatant was aspirated, and the released lactate dehydrogenase (LDH) was measured using the LDH Activity Assay Kit (Beyotime, China) according to the manufacturer's instructions, and served as a marker of cell death in the culture medium.

value: Brian

value: cortical  neurons

value: primary cultured cortical  neurons cells


title: Trauma3

status: Public on May 10 2024

type: SRA

country: China

library_source: transcriptomic

molecule: polyA RNA

organism: Mus musculus

tag: tissue

tag: cell line

tag: cell type


title: VSMC, WT, H3K18LA CUT&Tag

city: Shanghai

first: Wenqi

last: Ma

state: Shanghai

address: 227 Chongqing South Road, Huangpu District, Shanghai, China

institute: Shanghai Jiaotong University

library_source: genomic

data_processing:
*library strategy: CUT&Tag
All reads produced by CUT&Tag of NR4A3 and H3K18la were aligned to the Mouse reference genome (UCSC, mm10 assembly) using Bowtie2 with options: –local– very-sensitive-local–no-unal–no-mixed–no-discordant–phred33 -I 10 -X 700.
Sequence tags were aligned to the genome and then subsequently analyzed by MACS2 (version 2.2.7.1) to detect genomic regions enriched for multiple overlapping DNA fragments (peaks) that we considered to be putative binding sites.
We considered differential peaks with FDR lower than 5 % as statistically significant.
Visualization of peak distribution along genomic regions of interested genes was performed with IGV.
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes counts for each sample
Supplementary files format and content: bigwig, narrowpeak

molecule: genomic DNA

source_name: Aorta

growth_protocol: Cells were cultured in DMEM/F12 medium containing 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin, and 10 mM β-GP.

extract_protocol:
Total RNA was extracted from the cultured cell samples using TRIzol Reagent. RNA quality and quantity were measured using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system
Libraries were sequenced on Illumina Nova-PE150, and 150-bp paired-end reads were generated

treatment_protocol: Cells at approximately 80 % confluence were cultured in DMEM/F12 medium containing 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin, and 10 mM β-GP.

value: Aorta

value: VCMC

value: Primary

tag: genotype

value: WT

tag: treatment

value: β-GP


title: Trauma2


title: Spinal cord, Injury , P6981_1300

city: Solna

first: Christian

last: Göritz

address: Solnavägen 9

department: CMB

country: Sweden

institute: Karolinska Institutet

data_processing:
Clustering was done by 'cBot' and samples were sequenced on Illumina HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 1x51 setup using 'HiSeq SBS Kit v4' chemistry. The Bcl to FastQ conversion was performed using bcl2fastq from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+. Mus musculus, GRCm38 was used as reference genome.   We used a single-cell RNA sequencing snakemake workflow (“lts_workflows_sm_scrnaseq”) for alignment, quantification and quality control using resources as follows: The reads were aligned to the mouse (mm10) genome using STAR 2.5.3a (Dobin et al., 2013) and transcripts quantified in terms of Reads Per Kilobase of transcript per Million mapped reads (RPKM) using rpkmforgenes v1.0.1  (Ramsköld et al., 2009) and RSEM to estimate gene and isoform expression levels v1.2.28  (Li and Dewey, 2011). RSeQC V2.6.4  (Wang et al., 2012) Multiqc v0.9.1 were used for quality control. To filter out low quality libraries the following filtering criteria for quality control were applied: star_Uniquely mapped: < mean - 2 sd, gene_detection : > mean - 2 sd & < mean + 2 sd, count_gene : < mean - 2 sd, gene_correlation_max : < mean - 2 sd. Cells that failed for 2 of the criteria were filtered out (25 cells, 2.89% of 864, the total number of cells).
Assembly: mm10
Supplementary files format and content: rpkmforgenes_counts.tab
Supplementary files format and content: 170706_sampleinfo_scs.csv

molecule: total RNA

source_name: Spinal cord

extract_protocol:
Preparation of single-cell cell suspension and myelin removal was adapted from previously published protocols (Lovatt et al., 2007; Orre et al., 2014). In summary, the procedure was: 5 days after spinal cord crush injury, animals were euthanized by intraperitoneal injection of sodium pentobarbital (200mg/Kg, 100ul i.p., APL) and transcardially perfused with cold Hank's Balanced Salt Solution without calcium or magnesium (HBSS) (Invitrogen). The vertebral column was immediately removed to cold HBSS, the spinal cord was carefully dissected out of the vertebrae and the crush injury segment was separated from the uninjured spinal cord and treated separately hereafter. After removal of the outer meningeal layers, the tissue was dissociated mechanically with a razor blade on a glass petri dish and immediately transferred to cold HBSS. Followed by enzymatic digestion with papain at a final concentration of 8 U/mL with 80 Kunitz units/mL DNase I (Sigma-Aldrich, D4263) in Ca2+/Mg2+-free piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)/cysteine -based buffer, pH 7.4, for 40min at 37°C, 350rpm on a ThermoMixer® (Eppendorf). The tissue was carefully triturated and incubated for additional 5min. The cell suspension was passed through a 70μmCell Strainer (Corning), washed with Minimum Essential Media (MEM) with 1% bovine serum albumin (BSA) and spun down at 200g for 5min at 4°C. The cells were resuspended in MEM and centrifuged over a 90% Percoll gradient (GE Healthcare, 17-0891-01) at 250g for 15min at 4°C. Cells in the lipid layer and below were diluted 5 times in MEM with 1% BSA and spun in a 15mL tube at 250g for 10min at 4°C. All supernatant including the lipid layer were carefully removed and the pellet resuspended in cold MACS Buffer and magnetic Myelin removal beads (Miltenyi biotech, 130-096-433) and incubated for 15min at 4°C. The cells were washed and run over MACS MS columns (Miltenyi biotech, 130-042-201) on a magnetic stand according to the manufacturer’s instructions. The flow through cells were collected, spun down and resuspended in FACS buffer. The single cell suspensions derived from normal spinal cord and injury segments were subject to FACS (fluorescence activated cell sorting) into GLAST-CreER-tomato+/Pdgfrb-eGFP+ double positive and Pdgfrb-eGFP+ single cells, into 96-well plates with Cell lysis buffer (Clonetech, 635013) and frozen to -80°C. Cell sorting was performed on a FACSAria III Cell Sorter system (BD Biosciences). Singlet discrimination was performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H), dead cells were excluded by SYTOX Blue Dead Cell Stain (ThermoFisher Scientific, S34857).
Single cells were processed to cDNA libraries according to the Smart-Seq2 protocol  (Picelli et al. 2014). Tn5 tagmentation was performed using the Nextera XT DNA library Preparation Kit (Illumina, FC-131-1002) using dual indexes (i7 and i5).

value: Spinal cord

value: Perivascular cells

tag: Sex

value: female

tag: sorted population

value: EGFP+; tdTomato+


title: Spinal cord, Injury , P6981_1302


title: Spinal cord, Injury , P6981_1299


title: Spinal cord, Injury , P6981_1310


title: Tra_TGF3


title: Trauma1


title: Tra_TGF4


title: Spinal cord, Injury , P6981_1298


title: VSMC, NR4A3KO, H3K18LA CUT&Tag


title: Spinal cord, Injury , P6981_1297


title: Spinal cord, Injury , P6981_1294


title: Spinal cord, Injury , P6981_1296


title: Spinal cord, Injury , P6981_1295


title: Tra_TGF1


title: Control3


title: Control4


title: Control2


title: ChIPSeq 5bKI IL2 STAT5

city: Bethesda

first: Peng

last: Li

state: MD

address: 9000 Rockville Pike

department: NHLBI

country: USA

institute: NIH

data_processing:
Sequenced reads were aligned to the mm10 genome assembly using Bowtie 2.2.6 and Tophat 2.0.11
Aligned reads are converted to BAM files using samtools v0.1.8. BAM files are subsequently converted to BED files using bedtools v2.25.0. MACS is used to call peaks using the BED files. Unpublished python scripts were used to calculate RPKM (reads per kilobase per million reads) to normalize gene expression values for RNA-Seq data. R package edgeR was used to identify differentially expressed genes.
Assembly: mm10/GRCm38

source_name: spleen

growth_protocol: Splenic CD8+ T cells were purified using the EasySep Mouse CD8+ T cell Isolation Kit (STEMCell Technologies, 19853) and cultured in complete RPMI 1640 medium either without or with 500 IU/ml recombinant human IL-2 (Biological Resources Branch Preclinical Biologics Repository, NCI).

extract_protocol:
RNA-seq libraries were prepared using 100 ng of total RNA from splenic CD8+ T cells and the KAPA mRNA HyperPrep Kit (KR1352, KAPA Biosystems, Wilmington, MA), and each library was indexed using NEXflex DNA Barcodes-24 (BIOO Scientific, Austin, TX). Barcoded libraries were separated on 2% E-Gel, 250–400 bp fragments were purified, quantified on Qbit (Invitrogen), and equal amounts of each library were pooled and sequenced on an Illumina NovaSeq platform. For ChIP-seq libraries, freshly isolated splenic CD8+ T cells from Stat5a KI, Stat5b KI, and WT littermate mice were either not treated or treated with 500 IU/ml of IL-2 for 4 h, and cross-linked with 1% formaldehyde (methanol-free, Pierce, Rockford, IL) at room temperature for 10 min. After sonication at 30” ON and 30” OFF for 5 times using Bioruptor Pico (Diagenode), fragmented chromatin equivalent to 7 million cells were immunoprecipitated with control rabbit IgG (3900S, Cell Signaling Technology) or anti-STAT5A/B antibody (Ab194898, Abcam), and 20 μl of Magna ChIP Protein A + G Magnetic Beads (16–663, Millipore, Billerica MA). ChIP-Seq DNA libraries were prepared using KAPA Hyper Prep Kit (KR0961, KAPA Biosystems, Wilmington, MA) and NEXflex DNA Barcodes-24 (NOVA- 514103, BIOO Scientific, Austin, TX), and the indexed libraries were pooled and sequenced on an Illumina NovaSeq platform.
After quantification by Qubit (Invitrogen), barcoded samples were mixed and sequenced on an Illumina NovaSeq system.

treatment_protocol: The purified CD8+ T cells were cultured in the presence of 500 U/ml of recombinant human IL-2, and then collected at 0, 4, 24, and 48 hr. The cells were washed with PBS once, lysed in TriReagent (Zymo Research, Irvine, CA), and total RNA was isolated using Direct-zol RNA MicroPrep Kit (Zymo Research, Irvine, CA).

value: spleen

tag: strain

value: freshly isolated CD8+ T cells

tag: treatment group

value: 5bKI IL2 STAT5


title: Spinal cord, Injury , P6981_1293


title: Spinal cord, Injury , P6981_1301


title: Spinal cord, Injury , P6981_1291


title: ChIPSeq 5bKI Cont STAT5

value: 5bKI Cont STAT5


title: Spinal cord, Injury , P6981_1292


title: Spinal cord, Injury , P6981_1290


title: Control1


title: ChIPSeq 5bWT IL2 STAT5

value: 5bWT IL2 STAT5


title: Spinal cord, Injury , P6981_1287

value: EGFP+; tdTomato-


title: ChIPSeq 5aKI IL2 STAT5

value: 5aKI IL2 STAT5


title: Spinal cord, Injury , P6981_1285


title: ChIPSeq 5aKI Cont STAT5

value: 5aKI Cont STAT5


title: ChIPSeq 5bWT Cont STAT5

value: 5bWT Cont STAT5


title: ChIPSeq 5aWT Cont STAT5

value: 5aWT Cont STAT5


title: Spinal cord, Injury , P6981_1288


title: Spinal cord, Injury , P6981_1286


title: ChIPSeq 5aWT IL2 STAT5

value: 5aWT IL2 STAT5


title: Spinal cord, Injury , P6981_1284


title: ChIPSeq 5aWT IL2 IgG control

value: 5aWT IL2 IgG control


title: Tra_TGF2

value: Brian

value: cortical  neurons

value: primary cultured cortical  neurons cells


title: RNASeq STAT5B.48h.KI rep3


title: RNASeq STAT5B.48h.KI rep2


title: Spinal cord, Injury , P6981_1282

value: Spinal cord

value: Perivascular cells

value: female


title: RNASeq STAT5B.48h.KI rep1

value: spleen

value: freshly isolated CD8+ T cells


title: Spinal cord, Injury , P6981_1281


title: RNASeq STAT5B.48h.WT rep3


title: Spinal cord, Injury , P6981_1280


title: Spinal cord, Injury , P6981_1279


title: RNASeq STAT5B.48h.WT rep2


title: Spinal cord, Injury , P6981_1289

value: EGFP+; tdTomato+


title: Spinal cord, Injury , P6981_1278


title: Spinal cord, Injury , P6981_1277


title: RNASeq STAT5B.24h.KI rep3


title: RNASeq STAT5B.24h.KI rep2


title: RNASeq STAT5B.48h.WT rep1


title: RNASeq STAT5B.24h.KI rep1


title: Spinal cord, Injury , P6981_1275


title: Spinal cord, Injury , P6981_1274


title: RNASeq STAT5B.24h.WT rep3


title: RNASeq STAT5B.24h.WT rep2


title: Spinal cord, Injury , P6981_1273


title: Spinal cord, Injury , P6981_1271


title: Rat_9_con_distal

city: Aarhus

first: Hanne

middle: B

last: Moeller

address: Wilhelm Meyers Alle 3, building 1233

department: Dept. of Biomedicine

country: Denmark

institute: Aarhus University

data_processing:
HiSeq Control Software 2.2.58 RTA 1.18.64 bcl2fastq-1.8.4 software was used for basecalling.
Sequences are demultiplexed according to the 2x8 bp index code with 1 mismatch allowed. All reads are passed filter, i.e. reads have passed the default Illumina filter procedure (chastity filter).
BWA-MEM version 0.7.12-r1039 was used to map RNA reads to reference sequences (Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv:1303.3997v2[q-bio.GN]).
Normalization of reads was performed using Trimmed Mean of M values (TMM) normalization method with the edgeR-package version 3.16.2 in R.
Genome_build: rn6
Supplementary_files_format_and_content: GEN160929_M.profiling-table.txt contains a Matrix table with both the raw counts and CPM normalized counts for every gene and every sample

organism: Rattus norvegicus

source_name: distal colon

growth_protocol: Twelve male Wistar Munich rats were housed in standard cages with ad libitum access to water and standard rodent chow (cat. no. 1321, Altromin). Animals weighed between 284g and 347g, when they were put in metabolic cages.

extract_protocol:
After 10 days on the different diets rats were euthanized by cervical dislocation, their abdomens opened and the colons were dissected out and flushed clean off faeces with cold PBS.
Colons were divided into thirds and the middle section discarded. The proximal segment (initial 1/3) and the distal segment (last 1/3) were inverted, tied together at both ends and filled with Ca2+ free Ringer buffer with EDTA to form small sacs.
The colon sacs were put in separate 50 mL tubes with warm Ca2+ free Ringer buffer with EDTA and incubated at 37°C for 20 min. The tubes were shaken vigorously to release colonic epithelial cells (CECs) from the colon sacs. Colon sacs were then removed and the CEC suspensions were divided into two tubes and centrifuged at 4000 g for 3 min to pellet the cells. Supernatants were discarded and pellets were resuspended and homogenized in either 1 mL TriReagent™ (Ambion®) for RNA-analysis or in dissection buffer for protein analysis.
TriReagent samples were sonicated and RNA was extracted using the RiboPure™ RNA Purification kit (Ambion®) according to the manufacturer’s instructions. RNA yield was estimated based on A260 measurements by spectrophotometry using a NanoPhotometer® (Implen) with Elution Buffer from the RiboPure™ kit as blank.
Preparation of strand-specific cDNA libraries, RNA selection using polyA purification and single channel RNA sequencing was performed by Eurofins Genomics (Germany) using HiSeq SBS Kit v4 on a HiSeq 2500 (Illumina).

treatment_protocol:
Animals were housed at 22°C and with a 12:12 h light:dark cycle. During the experiment, the rats were housed in single-animal metabolic cages (Techniplast), where animal weight, water and food consumption as well as urine and faeces output were monitored daily.
For an initial three days, the rats were acclimatized to the metabolic cages with ad libitum access to water and standard rodent chow. Subsequently, six rats were switched to standard rodent chow mixed with 1% w/w sodium cholate (Sigma Aldrich).

value: Wistar Munich

value: distal colon

value: control

tag: gender

value: male


title: Spinal cord, Injury , P6981_1283

value: EGFP+; tdTomato-


title: Spinal cord, Injury , P6981_1270


title: Spinal cord, Injury , P6981_1269


title: Rat_8_BA_distal

value: Rodent chow mixed with 1% w/w sodium cholate


title: RNASeq STAT5B.4h.KI rep3


title: RNASeq STAT5B.24h.WT rep1


title: Rat_7_BA_distal


title: RNASeq STAT5B.4h.KI rep2


title: Spinal cord, Injury , P6981_1268


title: Spinal cord, Injury , P6981_1267


title: Spinal cord, Injury , P6981_1276


title: Spinal cord, Injury , P6981_1265


title: Rat_6_con_distal


title: Spinal cord, Injury , P6981_1266


title: RNASeq STAT5B.4h.KI rep1


title: Spinal cord, Injury , P6981_1264


title: Spinal cord, Injury , P6981_1263


title: Rat_5_con_distal


title: Rat_4_BA_distal


title: Spinal cord, Injury , P6981_1272


title: Spinal cord, Injury , P6981_1261


title: RNASeq STAT5B.4h.WT rep2


title: Spinal cord, Injury , P6981_1262


title: Spinal cord, Injury , P6981_1260


title: Spinal cord, Injury , P6981_1259


title: Spinal cord, Injury , P6981_1258


title: Spinal cord, Injury , P6981_1256


title: Rat_1_con_distal


title: Spinal cord, Injury , P6981_1257


title: RNASeq STAT5B.0h.KI rep2


title: Spinal cord, Injury , P6981_1254


title: RNASeq STAT5B.0h.KI rep1


title: Rat_11_BA_distal


title: Spinal cord, Injury , P6981_1255


title: Spinal cord, Injury , P6981_1252


title: RNASeq STAT5B.4h.WT rep3


title: Spinal cord, Injury , P6981_1249


title: Spinal cord, Injury , P6981_1251


title: Spinal cord, Injury , P6981_1253


title: RNASeq STAT5B.0h.WT rep2


title: Spinal cord, Injury , P6981_1250


title: Spinal cord, Injury , P6981_1246


title: RNASeq STAT5B.0h.WT rep1


title: Rat_3_BA_distal


title: Spinal cord, Injury , P6981_1248


title: RNASeq STAT5B.4h.WT rep1


title: 913913

city: Paris

first: Bastien

last: Leclercq

state: Ile-de-France

address: 15 Rue de l'École de Médecine

country: France

institute: INSERM

data_processing:
Quality of raw data was evaluated with FastQC. Poor quality sequences and adapters were trimmed or removed with fastp, using default parameters, to retain only good quality paired reads
Illumina DRAGEN bio-IT Plateform (v3.8.4) was used for mapping on rn7 ratus norvegicus reference genome
Library orientation, library composition and coverage along transcripts were checked with Picard tools
The transcriptomic data were analyzed on the online platform of the Paris Brain institute (quby.icm-institute.org). Firstly, differential expression analysis using DESeq2 method was assessed to determine the differentially expressed genes (adjusted P-value (pFDR), FDR threshold set at 0.05, log2 fold-change threshold set at 0).
Assembly: Illumina DRAGEN bio-IT Plateform (v3.8.4) was used for mapping on rn7 ratus norvegicus reference genome
Supplementary files format and content: List of regulated genes DESeq2, excel format
Supplementary files format and content: List of regulated genes DESeq2, excel format

source_name: Neuroretina

extract_protocol:
RNA was extracted using the RNeasy Mini Kit (Qiagen) and processed as per manufacturer’s instructions.
Illumina DRAGEN bio-IT Plateform (v3.8.4) was used for mapping on rn7 ratus norvegicus reference genome
Library orientation, library composition and coverage along transcripts were checked with Picard tools

treatment_protocol: Six months old female P1.hMR rats (n=5) and their age and sex-matched wild-type littermates (n=2) were euthanized by intraperitoneal injection of EUTHASOL VET (300 mg/kg, Dechra, Northwich, UK) and the eyes were removed and quickly dissected on ice. Neuroretinas were isolated and frozen in liquid nitrogen

value: Neuroretina

value: wild-type

value: untreated


title: 905946


title: 903260


title: 835531


title: Spinal cord, Injury , P6981_1247


title: Spinal cord, Injury , P6981_1245


title: 835181


title: Rat_2_con_distal


title: RNASeq STAT5A.48h.KI rep3


title: RNASeq STAT5B.0h.KI rep3


title: Spinal cord, Injury , P6981_1243


title: RNASeq STAT5B.0h.WT rep3


title: Spinal cord, Injury , P6981_1244


title: Spinal cord, Injury , P6981_1242


title: Spinal cord, Injury , P6981_1240


title: Spinal cord, Injury , P6981_1239


title: RNASeq STAT5A.48h.KI rep1


title: Spinal cord, Injury , P6981_1241


title: 91740


title: RNASeq STAT5A.48h.WT rep3


title: Spinal cord, Injury , P6981_1234


title: RNA-Seq MMI rep1

city: Moscow

first: Nickolai

last: Tchurikov

address: 32, Vavilova str.

department: Department of Epigenetic Mechanisms of Gene Expression Regulation

country: Russia

institute: Engelhardt Institute of Molecular Biology

description: Replica 1

data_processing:
Base calling and quality control were performed in real time with standard Illumina analysis pipeline using a phiX control.
Sequenced reads were trimmed from low quality and/or short sequences by Trimmomatic v. 0.39 using the following options:  LEADING:22 TRAILING:22 SLIDINGWINDOW:4:26 MINLEN:20
Trimmed reads were mapped to H.sapiens genome (hg38/GRCh38 Ensembl v.106) by STAR aligner 2.7.5c with hg38 Ensembl genome map v.106 http://ftp.ensembl.org/pub/release-106/gtf/homo_sapiens/Homo_sapiens.GRCh38.106.gtf.gz. Alignment options were the following:  --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --quantMode TranscriptomeSAM
Alignment files were separated by samtools 1.15 view -F 0x10 to get forward strand alignments and by samtools 1.15 view -f 0x10 to get reverse strand alignments. Then bamCoverage (deepTools 3.5.1) tool was used to obtain normalized expression profiles. The bamCoverage options list is the following: --binSize 10 --effectiveGenomeSize 2913022398 --skipNAs --normalizeUsing RPKM --exactScaling. The obtained profiles were averaged between replicates by WiggleTools v1.2.11 and converted to bigWig format by bedGraphToBigWig tool from UCSC genome tools.
Trimmed reads were quantified to H. sapiens hg38 GRCh38 Ensembl v.106 genome with Ensembl genome map v.106 by RSEM 1.3.1 using the following options: --fragment-length-mean 255 --star --calc-ci --ci-memory 30720. This operation was performed for both replicates separately. The results were averaged by in-house R script.
Assembly: Homo sapiens hg38/GRCh38 Ensembl release 106 genome build. Homo sapiens hg38/GRCh38 Ensembl annotation v106 in GTF format.
Supplementary files format and content: bigWig files contain normalized (RPKM, reads per kilobase million) expression signal genomewide for each strand. Results files contain quantified expression values in TPM (Transcripts Per Million) and FPKM (Fragments Per Kilobase Million) for each gene averaged between replicates.

organism: Homo sapiens

source_name: melanoma cells

growth_protocol: The Mel Z melanoma cells initially were propagated on plastic surface in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and 0.1% gentamicin sulphate at 37°C under a humidified atmosphere containing 5% CO2 up to 10 million of cells with 70–75% confluency. Then the cells were divided into three equal samples and seeded either on plastic, or on Matrigel, or on Matrigel in the presence of non-cytotoxic concentration of inhibitor (LCS1269, 10-6 M) in the same medium. The growth was performed under the same conditions for 20 h, and then the cells were used for RNA isolation. This sample corresponds to cells grown for 20 h on Matrigel in the presence of the inhibitor (MMI).

extract_protocol:
Total RNA was extracted from cell lysed with Trisol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA Quality was checked with  BioAnalyser and  RNA 6000 Nano Kit(Agilent). Poly(A)+ RNA was isolated with Dynabeads® mRNA Purification Kit (Ambion).
Poly(A)+ RNA was isolated with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length.

value: Mel Z melanoma cells

value: skin

value: melanocytes


title: Spinal cord, Injury , P6981_1236

value: Spinal cord

value: Perivascular cells


title: RNA-Seq MM rep2

description: Replica 2

data_processing:
Base calling and quality control were performed in real time with standard Illumina analysis pipeline using a phiX control.
Sequenced reads were trimmed from low quality and/or short sequences by trimmomatic v. 0.39 using the following options:  LEADING:22 TRAILING:22 SLIDINGWINDOW:4:26 MINLEN:20
Trimmed reads were mapped to H.sapiens genome (hg38/GRCh38 Ensembl v.106) by STAR aligner 2.7.5c with hg38 Ensembl genome map v.106 http://ftp.ensembl.org/pub/release-106/gtf/homo_sapiens/Homo_sapiens.GRCh38.106.gtf.gz. Alignment options were the following:  --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --quantMode TranscriptomeSAM
Alignment files were separated by samtools 1.15 view -F 0x10 to get forward strand alignments and by samtools 1.15 view -f 0x10 to get reverse strand alignments. Then bamCoverage (deepTools 3.5.1) tool was applied to obtain normalized expression profiles. The bamCoverage options list is the following: --binSize 10 --effectiveGenomeSize 2913022398 --skipNAs --normalizeUsing RPKM --exactScaling. The obtained profiles were averaged between replicates by WiggleTools v1.2.11 and converted to bigWig format by bedGraphToBigWig tool from UCSC genome tools.
Trimmed reads were quantified to H. sapiens hg38 GRCh38 Ensembl v.106 genome with Ensembl genome map v.106 by RSEM 1.3.1 using the following options: --fragment-length-mean 255 --star --calc-ci --ci-memory 30720. This operation was performed for both replicates separately. The results were averaged by in-house R script.
Assembly: Homo sapiens hg38/GRCh38 Ensembl release 106 genome build. Homo sapiens hg38/GRCh38 Ensembl annotation v106 in GTF format.
Supplementary files format and content: bigWig files contain normalized (RPKM, reads per kilobase million) expression signal genomewide for each strand. Results files contain quantified expression values in TPM (Transcripts Per Million) and FPKM (Fragments Per Kilobase Million) for each gene averaged between replicates.

growth_protocol: The Mel Z melanoma cells initially were propagated on plastic surface in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and 0.1% gentamicin sulphate at 37°C under a humidified atmosphere containing 5% CO2 up to 10 million of cells with 70–75% confluency. Then the cells were divided into three equal samples and seeded either on plastic, or on Matrigel, or on Matrigel in the presence of non-cytotoxic concentration of inhibitor (LCS1269, 10-6 M) in the same medium. The growth was performed under the same conditions for 20 h, and then the cells were used for RNA isolation. This sample corresponds to cells grown for 20 h on Matrigel (MM).


title: Spinal cord, Injury , P6981_1235

value: female


title: RNA-Seq MMI rep2

description: Replica 2


title: Spinal cord, Injury , P6981_1233


title: RNASeq STAT5A.48h.WT rep2

value: spleen

value: freshly isolated CD8+ T cells


title: RNASeq STAT5A.48h.KI rep2


title: RNASeq STAT5A.48h.WT rep1


title: Spinal cord, Injury , P6981_1232


title: Spinal cord, Injury , P6981_1231


title: 602125


title: Spinal cord, Injury , P6981_1229


title: RNASeq STAT5A.24h.KI rep2


title: Spinal cord, Injury , P6981_1228


title: Spinal cord, Injury , P6981_1224


title: Spinal cord, Injury , P6981_1238


title: RNASeq STAT5A.24h.KI rep1


title: Spinal cord, Injury , P6981_1230


title: Spinal cord, Injury , P6981_1225


title: Spinal cord, Injury , P6981_1223


title: RNASeq STAT5A.24h.KI rep3


title: RNASeq STAT5A.24h.WT rep1


title: Spinal cord, Injury , P6981_1226


title: Spinal cord, Injury , P6981_1221


title: Spinal cord, Injury , P6981_1222


title: Spinal cord, Injury , P6981_1227


title: Spinal cord, Injury , P6981_1237


title: RNA-Seq MM rep1

description: Replica 1


title: Spinal cord, Injury , P6981_1220


title: RNASeq STAT5A.4h.KI rep3


title: RNASeq STAT5A.4h.KI rep2


title: Spinal cord, Injury , P6981_1218


title: RNASeq STAT5A.4h.KI rep1


title: Spinal cord, Injury , P6981_1216


title: Spinal cord, Injury , P6981_1217


title: RNASeq STAT5A.24h.WT rep3


title: Spinal cord, Injury , P6981_1215


title: RNASeq STAT5A.4h.WT rep3


title: Spinal cord, Injury , P6981_1214


title: Spinal cord, Injury , P6981_1213


title: RNASeq STAT5A.4h.WT rep2


title: Spinal cord, Injury , P6981_1212


title: RNASeq STAT5A.24h.WT rep2


title: Spinal cord, Injury , P6981_1210


title: RNASeq STAT5A.4h.WT rep1


title: Spinal cord, Injury , P6981_1209


title: Spinal cord, Injury , P6981_1204


title: Spinal cord, Injury , P6981_1208


title: RNASeq STAT5A.0h.KI rep2


title: RNASeq STAT5A.0h.KI rep1


title: Spinal cord, Injury , P6981_1203


title: Spinal cord, Injury , P6981_1206


title: Spinal cord, Injury , P6981_1207


title: Spinal cord, Injury , P6981_1205


title: RNASeq STAT5A.0h.WT rep3


title: Spinal cord, Injury , P6981_1219


title: RNASeq STAT5A.0h.WT rep2


title: Spinal cord, Injury , P6981_1200


title: Spinal cord, Injury , P6981_1202


title: Spinal cord, Injury , P6981_1201


title: Spinal cord, Injury , P6981_1199


title: RNASeq STAT5A.0h.WT rep1


title: Spinal cord, Injury , P6981_1197


title: WT

city: Singapore

first: Yingying

last: Zeng

state: --- Select a state ---

address: 61 Biopolis Drive

country: Singapore

institute: Institute of Molecular and Cell Biology

data_processing:
Hi-C data Read pairs were aligned to mm9 genome with BWA-MEM program. Duplicates removing, .pair and bam files generation step were done by Pairtools pipeline. .hic files were generated using juicer tools ‘pre’ option.
Assembly: mm9
Supplementary files format and content: .hic

source_name: Primary mouse neural stem cells

extract_protocol:
Samples were prepared according to the Dovetail Topolink kit protocol version 1.2 (Cantata Bio). Briefly, 1 million cells were crosslinked with disuccinimidyl glutarate and formaldehyde. Next, the nuclei were extracted and the chromatin was enzymatically fragmented. The profile of chromatin fragmentation was quality-controlled by Bioanalyzer DNA high sensitivity assay (Agilent Technologies). Following this, 3ng of fragmented chromatin was proximity ligated on chromatin capture beads.
The resulting chromatin was subjected to library preparation mix 1 and 2, then reverse crosslinked and quantified using Qubit dsDNA HS kit (Thermo Fischer). The DNA yield was split evenly into 4 reactions, and each was prepared into dual-indexed HiC library using library prep mix 3 and 4.

value: Primary mouse neural stem cells

value: NSC

value: WT


title: Spinal cord, Injury , P6981_1211


title: Spinal cord, Injury , P6981_1195


title: PHF2_KO


title: Spinal cord, Injury , P6981_1196


title: Spinal cord, Injury , P6981_1194


title: RNASeq STAT5A.0h.KI rep3


title: Spinal cord, Injury , P6981_1193


title: Tcell_depletion_biological_replicate_3

city: Munich

first: Ruoqing

last: Feng

address: Feodor-Lynen-Strasse 17

country: Germany

institute: German Center for Neurodegenerative Diseases (DZNE)

library_source: transcriptomic single cell

data_processing:
The raw sequencing data of each library was processed by Cell Ranger (10X Genomics) 3.0 to generate the gene-by-cell unique molecular identifier (UMI) count matrix
Assembly: GRCm38
Supplementary files format and content: sparse matrix h5

source_name: Brain

extract_protocol:
For 10X genomic experiments, mice were deeply anesthetized and perfused with cold PBS. Each brain was removed and individually microdissected under a dissection microscope, wherein the cortex and hippocampus were isolated from the brain and the attached choroid plexus was removed. Cells were isolated with a previously established isolation protocol 27using gentleMACS with the Neural Tissue Dissociation Kit (Papain; Miltenyi Biotec) and actinomycin D (Act-D, Sigma-Aldrich, catalog no. A1410) at a final concentration of 45 mM. After dissociation, myelin debris were removed from the cell suspension using Myelin Removal Beads II (Miltenyi Biotec). The cells were resuspended in 0.04% BSA+PBS and cells were counted using automated cell counter (TC20 Bio-Rad) before loading to Chromium Controller.
Chromium Single Cell 3′ Library & Gel Bead Kit v.3.1 (10X Genomics)
Single-cell suspensions were loaded on to the Chromium Single Cell Controller using the Chromium Single Cell 3′ Library & Gel Bead Kit v.3.1 (10X Genomics) chemistry following the manufacturer’s instructions. Sample processing and library preparation were performed according to the manufacturer’s instructions using AMPure beads (Beckman-Coulter). Libraries were sequenced on the DNBSEQ Sequencing System (Dresden).

value: Brain


title: Spinal cord, Uninjured, P6981_1191

value: EGFP+; tdTomato+


title: Spinal cord, Uninjured, P6981_1192


title: Tcell_depletion_biological_replicate_2


title: Spinal cord, Uninjured, P6981_1189


title: Spinal cord, Uninjured, P6981_1190


title: Control_biological_replicate_3

value: Control


title: Spinal cord, Uninjured, P6981_1187


title: Spinal cord, Uninjured, P6981_1188


title: Spinal cord, Uninjured, P6981_1185


title: Control_biological_replicate_2


title: Spinal cord, Uninjured, P6981_1186


title: Control_biological_replicate_1


title: Spinal cord, Uninjured, P6981_1183


title: Spinal cord, Uninjured, P6981_1184


title: Spinal cord, Uninjured, P6981_1179


title: Spinal cord, Uninjured, P6981_1182


title: Spinal cord, Uninjured, P6981_1181


title: Spinal cord, Uninjured, P6981_1177


title: Spinal cord, Uninjured, P6981_1180


title: Spinal cord, Uninjured, P6981_1174


title: Spinal cord, Injury , P6981_1198

value: EGFP+; tdTomato-


title: Spinal cord, Uninjured, P6981_1178


title: Spinal cord, Uninjured, P6981_1176


title: Spinal cord, Uninjured, P6981_1175


title: Tcell_depletion_biological_replicate_1


title: Spinal cord, Uninjured, P6981_1173


title: Spinal cord, Uninjured, P6981_1172


title: Spinal cord, Uninjured, P6981_1171


title: Spinal cord, Uninjured, P6981_1170


title: Spinal cord, Uninjured, P6981_1169


title: Early Gastric Carcinoma (ECG), C6A, miRNA

city: Chengdu

first: Jinlin

last: Yang

state: Sichuan Province

address: Guoxue Lane, Wuhou District

institute: Department of Gastroenterology and Hepatology, West China Hospital of Sichuan University

data_processing:
The raw data were quality-controlled using fastp software to obtain high-quality clean data. Clean data of miRNAs were compared and analyzed using the QuickMIRSeq tools.
Assembly: hg18
Supplementary files format and content: tab-delimited text files include counts values for each Sample

source_name: cancer tissue

extract_protocol:
The miRNA libraries were constructed and sequenced on the BGISEQ-500 platform with the SE50 strategy, respectively. Based on Sequencing By Synthesis (SBS) technology.
The cDNA library was sequenced by DNBSEQ high-throughput sequencing platform, which produces a large amount of high-quality data, known as raw data.

value: cancer tissue


title: pcPrF-M31

type: RNA

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: 10-1 tebiro 6-chome

country: Japan

institute: Toray Industries,Inc.

hyb_protocol: Hybridized for 16 h at 32 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..

scan_protocol: 3D-Gene Scanner (Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).

data_processing: The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals.

source_name: primary cultured prostate fibroblasts

label_protocol: Extracted total RNA was labeled with Cy5 using the 3D-Gene miRNA labeling kit (Toray, Kamakura, Japan)

growth_protocol: Fibroblasts (1.0 × 105 cells/well) were seeded in 6-well plates with SCBM Bullet Kit (Lonza Group Ltd.) and were cultured for 72 h.

extract_protocol: RNA samples were extracted from cells using miRNesy mini kit (Qiagen).

treatment_protocol: The culture medium was replaced with phenol red (-) RPMI-1640 supplemented with 1% CS-FBS containing DHT (0.1 nM) and cultured for 48 h.

value: prostate cancer

value: Male

tag: Stage

tag: gleason score


title: Spinal cord, Uninjured, P6981_1168


title: Early Gastric Carcinoma (ECG), C7A, miRNA


title: PrSC

source_name: human prostate stromal cells

value: prostate


title: pcPrF-M28


title: pcPrF-M5


title: Early Gastric Carcinoma (ECG), C1, miRNA


title: Chronic Atrophic Gastritis (CAG), B30A, miRNA

source_name: atrophic gastric mucosa

value: atrophic gastric mucosa


title: Chronic Atrophic Gastritis (CAG), B27A, miRNA


title: Spinal cord, Uninjured, P6981_1165

value: Spinal cord


title: Spinal cord, Uninjured, P6981_1166

value: Perivascular cells


title: Spinal cord, Uninjured, P6981_1167

value: female


title: Spinal cord, Uninjured, P6981_1164


title: Spinal cord, Uninjured, P6981_1163


title: Spinal cord, Uninjured, P6981_1161


title: Spinal cord, Uninjured, P6981_1162


title: Chronic Atrophic Gastritis (CAG), B12, miRNA


title: Spinal cord, Uninjured, P6981_1160


title: Spinal cord, Uninjured, P6981_1158


title: Chronic Atrophic Gastritis (CAG), B11, miRNA


title: Spinal cord, Uninjured, P6981_1159


title: Spinal cord, Uninjured, P6981_1157


title: Chronic Atrophic Gastritis (CAG), B1, miRNA


title: Spinal cord, Uninjured, P6981_1156


title: Spinal cord, Uninjured, P6981_1155


title: Chronic Non-atrophic Gastritis (CNAG), A30A, miRNA

source_name: non-atrophic gastric mucosa

value: non-atrophic gastric mucosa


title: Spinal cord, Uninjured, P6981_1153


title: Spinal cord, Uninjured, P6981_1154


title: Spinal cord, Uninjured, P6981_1152


title: Chronic Non-atrophic Gastritis (CNAG), A27A, miRNA


title: Spinal cord, Uninjured, P6981_1151


title: Spinal cord, Uninjured, P6981_1147


title: Spinal cord, Uninjured, P6981_1150


title: Chronic Non-atrophic Gastritis (CNAG), A20, miRNA


title: Chronic Non-atrophic Gastritis (CNAG), A12, miRNA


title: Spinal cord, Uninjured, P6981_1144


title: Spinal cord, Uninjured, P6981_1148


title: Chronic Non-atrophic Gastritis (CNAG), A11, miRNA


title: Chronic Atrophic Gastritis (CAG), B20, miRNA


title: Spinal cord, Uninjured, P6981_1146


title: Spinal cord, Uninjured, P6981_1143


title: Early Gastric Carcinoma (ECG), C6A, lncRNA

data_processing:
The raw data were quality-controlled using fastp software to obtain high-quality clean data. Clean data of lncRNA were compared to the human reference genome using HISAT2 software.
Assembly: hg18
Supplementary files format and content: tab-delimited text files include counts values for each Sample

extract_protocol:
The lncRNA libraries were constructed and sequenced on the BGISEQ-500 platform with the PE100 strategy, respectively. Based on Sequencing By Synthesis (SBS) technology.
The cDNA library was sequenced by DNBSEQ high-throughput sequencing platform, which produces a large amount of high-quality data, known as raw data.


title: Spinal cord, Uninjured, P6981_1145


title: Early Gastric Carcinoma (ECG), C7A, lncRNA


title: Spinal cord, Uninjured, P6981_1141


title: Chronic Atrophic Gastritis (CAG), B30A, lncRNA


title: Early Gastric Carcinoma (ECG), C1, lncRNA


title: Spinal cord, Uninjured, P6981_1139


title: Spinal cord, Uninjured, P6981_1140


title: Spinal cord, Uninjured, P6981_1138


title: Spinal cord, Uninjured, P6981_1137


title: Spinal cord, Uninjured, P6981_1135


title: Chronic Atrophic Gastritis (CAG), B27A, lncRNA


title: Spinal cord, Uninjured, P6981_1149


title: Spinal cord, Uninjured, P6981_1136


title: Spinal cord, Uninjured, P6981_1133


title: Spinal cord, Uninjured, P6981_1134


title: Chronic Atrophic Gastritis (CAG), B20, lncRNA


title: Spinal cord, Uninjured, P6981_1132


title: Chronic Atrophic Gastritis (CAG), B12, lncRNA


title: Spinal cord, Uninjured, P6981_1129


title: Spinal cord, Uninjured, P6981_1131


title: Spinal cord, Uninjured, P6981_1142


title: Chronic Atrophic Gastritis (CAG), B1, lncRNA


title: Spinal cord, Uninjured, P6981_1130


title: Chronic Atrophic Gastritis (CAG), B11, lncRNA


title: Spinal cord, Uninjured, P6981_1127


title: Chronic Non-atrophic Gastritis (CNAG), A30A, lncRNA


title: Spinal cord, Uninjured, P6981_1124


title: Spinal cord, Uninjured, P6981_1123


title: Spinal cord, Uninjured, P6981_1128


title: Chronic Non-atrophic Gastritis (CNAG), A20, lncRNA


title: Spinal cord, Uninjured, P6981_1122


title: Spinal cord, Uninjured, P6981_1125


title: Chronic Non-atrophic Gastritis (CNAG), A27A, lncRNA


title: Spinal cord, Uninjured, P6981_1126


title: Spinal cord, Uninjured, P6981_1121


title: Spinal cord, Uninjured, P6981_1119


title: Chronic Non-atrophic Gastritis (CNAG), A11, lncRNA


title: Spinal cord, Uninjured, P6981_1117


title: Chronic Non-atrophic Gastritis (CNAG), A12, lncRNA


title: Spinal cord, Uninjured, P6981_1116


title: Spinal cord, Uninjured, P6981_1115


title: Spinal cord, Uninjured, P6981_1114


title: Spinal cord, Uninjured, P6981_1113


title: Spinal cord, Uninjured, P6981_1112


title: Spinal cord, Uninjured, P6981_1111


title: Spinal cord, Uninjured, P6981_1110


title: Spinal cord, Uninjured, P6981_1104


title: Spinal cord, Uninjured, P6981_1107


title: Spinal cord, Uninjured, P6981_1105


title: Spinal cord, Uninjured, P6981_1108


title: Spinal cord, Uninjured, P6981_1109


title: Spinal cord, Uninjured, P6981_1106


title: Spinal cord, Uninjured, P6981_1120


title: Spinal cord, Uninjured, P6981_1101


title: Spinal cord, Uninjured, P6981_1102


title: Spinal cord, Uninjured, P6981_1100


title: Spinal cord, Uninjured, P6981_1098


title: Spinal cord, Uninjured, P6981_1099


title: Spinal cord, Uninjured, P6981_1095


title: Spinal cord, Uninjured, P6981_1096


title: Spinal cord, Uninjured, P6981_1092


title: Spinal cord, Uninjured, P6981_1097


title: Spinal cord, Uninjured, P6981_1093


title: Spinal cord, Uninjured, P6981_1094


title: Spinal cord, Uninjured, P6981_1091


title: Spinal cord, Uninjured, P6981_1090


title: Spinal cord, Uninjured, P6981_1089


title: Spinal cord, Uninjured, P6981_1088


title: Spinal cord, Uninjured, P6981_1086


title: Spinal cord, Uninjured, P6981_1118


title: Spinal cord, Uninjured, P6981_1084


title: Spinal cord, Uninjured, P6981_1087


title: Spinal cord, Uninjured, P6981_1103


title: Spinal cord, Uninjured, P6981_1079


title: Spinal cord, Uninjured, P6981_1080


title: Spinal cord, Uninjured, P6981_1082


title: Spinal cord, Uninjured, P6981_1081


title: Spinal cord, Uninjured, P6981_1078


title: Spinal cord, Uninjured, P6981_1077


title: Spinal cord, Uninjured, P6981_1076


title: Spinal cord, Uninjured, P6981_1075


title: Spinal cord, Uninjured, P6981_1073


title: Spinal cord, Uninjured, P6981_1072


title: Spinal cord, Uninjured, P6981_1070


title: Spinal cord, Uninjured, P6981_1068


title: Spinal cord, Uninjured, P6981_1069


title: Spinal cord, Uninjured, P6981_1071


title: Spinal cord, Uninjured, P6981_1074


title: Spinal cord, Uninjured, P6981_1066


title: Spinal cord, Uninjured, P6981_1064


title: Spinal cord, Uninjured, P6981_1067


title: Spinal cord, Uninjured, P6981_1085


title: Spinal cord, Uninjured, P6981_1065


title: Spinal cord, Uninjured, P6981_1062


title: Spinal cord, Uninjured, P6981_1061


title: Spinal cord, Uninjured, P6981_1058


title: Spinal cord, Uninjured, P6981_1063


title: Spinal cord, Uninjured, P6981_1083


title: Spinal cord, Uninjured, P6981_1059


title: Spinal cord, Uninjured, P6981_1060


title: Spinal cord, Uninjured, P6981_1055


title: Spinal cord, Uninjured, P6981_1054


title: Spinal cord, Uninjured, P6981_1049


title: Spinal cord, Uninjured, P6981_1052


title: Spinal cord, Uninjured, P6981_1048


title: Spinal cord, Uninjured, P6981_1051


title: PROseq_Zfp574_6h_AID_depletion_rep2

city: Vienna

first: Alexander

last: Stark

address: Campus-Vienna-Biocenter 1

country: Austria

institute: The Research Institute of Molecular Pathology (IMP)

description:
PROseq_counts_Zfp574_0h_ctl_6h_depletion.txt
PROseq_DESeq2_Zfp574_6h_depletion_vs_0h_ctl.txt

data_processing:
Before mapping, the UMI was separated, and the Illumina adapters were trimmed using cutadapt v.1.18. Only reads with a length greater than 10 bp were then mapped using Bowtie v.1.2.2 26, initially to the mm10 version of the mouse genome. The mapping allowed for up to 2 mismatches and reported only the best alignment (-m 1 --best --strata) for each read. To ensure the counting of unique nascent RNA molecules, reads that mapped to the same genomic location were collapsed based on their UMIs, allowing for up to 1 mismatch. To create the PRO-seq coverage signal with the exact positions of RNA pol II molecules, only the first nucleotide of each read (i.e. the 3’ end of nascent transcripts) was considered and the strand swapped to match the transcription direction. A non-redundant CAGE-corrected gene set was used to count the number of UMI-collapesed 1nt-long mapped PRO-seq reads that overlap them.
Differential analysis was performed using DESeq227 (v.1.22.2).
Assembly: mm10
Supplementary files format and content: PROseq_counts_Zfp574_0h_ctl_6h_depletion.txt contain raw counts for all conditions and replicates.
Supplementary files format and content: PROseq_DESeq2_Zfp574_6h_depletion_vs_0h_ctl.txt contains classical DESeq2 output (see DESeq2 documentation for further details about columns content)
Library strategy: PRO-seq

source_name: mESC

growth_protocol: All mouse experiments presented here were carried out in diploid mouse embryonic stem cells (mESCs) that were derived from originally haploid HMSc2 termed AN3-128. The mESCs were cultivated without feeders in high-glucose-DMEM (Sigma-Aldrich) supplemented with 13.5% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 1x Penicillin-Streptomycin (Sigma-Aldrich), 1x MEM non-essential amino acid solution (Gibco), 1mM sodium pyruvate (Sigma-Aldrich), 50 mM β-mercaptoethanol (Merck) and in-house produced recombinant LIF.

extract_protocol:
PRO-seq protocol was performed as in Serebreni, L. et al. Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns. EMBO J 42(10), e113519 (2023) with a single modification: the nuclear run-on was performed at 37 °C for 3 min.
PRO-seq libraries were sequenced in  paired-end mode with 36-bp read length. To eliminate PCR duplicates, an 8-bp long unique molecular identifier (UMI) was incorporated at the 5′ end of the reads during the sample processing.

treatment_protocol: For each condition, 1x10^7 AID-Zfp574 cells were collected and nuclei were isolated after 6h of 500uM IAA treatment or no treatment (two biological replicates per condition). Spike-in control (S2 Drosophila cells; 1% of mESC cells) were added at the level of nuclei permeabilization step.

value: mESC

value: AID-Zfp574; N-terminal in OsTir1 parental cell line (FRT-EF1a-ARF16-HA-P2A-OsTiR1-noXhoI-3xmYC-T2A-mCherry-SV40polyA-F3)


title: Spinal cord, Uninjured, P6981_1053


title: Spinal cord, Uninjured, P6981_1045


title: PROseq_Zfp574_0h_AID_ctl_rep2


title: Spinal cord, Uninjured, P6981_1050


title: PROseq_Zfp574_0h_AID_ctl_rep1


title: PROseq_Zfp574_6h_AID_depletion_rep1


title: Spinal cord, Uninjured, P6981_1047


title: Spinal cord, Uninjured, P6981_1046


title: CUTNRUN_Zfp574_aV5_parental_ctl_cell_line_rep2

description: CUTNRUN_Zfp574_aV5_parental_ctl_cell_line_merged.bw

data_processing:
Single-end 50-bp long reads were mapped to the mm10 genome using bowtie v.0.12.9, allowing up to 3 mismatches and only uniquely mapping reads were retained.
Peaks were called for each individual replicate, as well as for the combined replicates against their respective input, using Macs2 v.2.1.2.1, with following settings: -f BEDPE -g mm -B --nomodel --extsize 300 --SPMR. The Macs2 generated BedGraph files that contain normalized coverage were converted into BigWig using bedGraphToBigWig. Given the high correlation between two replicates (PCC of 0.613 at a common set of peaks), only the merged sample was used for assigning bound genes if the peak was localized within +- 500 bp around the gene transcription start sites.
Assembly: mm10
Supplementary files format and content: For each condition and each replicate, individual .bw files can be used to visualize CUT&RUN signal using a genome browser (e.g. IGV) or to quantify the signal
Supplementary files format and content: For each condition, merged .bw file corresponds to the merge of the two biological replicates
Supplementary files format and content: For each replicate, individual .narrowPeak files contain MACS2 peaks
Supplementary files format and content: The merged .narrowPeak file contains MACS2 peaks called from merged samples
Library strategy: CUT&RUN

extract_protocol:
For each biological replicate, 1x10^6 cells from the AID-Zfp574 cell line or the Tir1 parental cell line were used. The Tir1 parental cell line is used as Input.
The protocol was performed as in Hendy, O. et al. Developmental and housekeeping transcriptional programs in Drosophila require distinct chromatin remodelers. Mol Cell 82(19), 3598-3612.E7 (2022) with a V5-tag antibody (Thermo Fisher, R960-25) that was added to a final dilution of 1:100.

value: OsTir1 parental cell line (FRT-EF1a-ARF16-HA-P2A-OsTiR1-noXhoI-3xmYC-T2A-mCherry-SV40polyA-F3)

tag: antibody


title: Spinal cord, Uninjured, P6981_1043


title: Spinal cord, Uninjured, P6981_1056


title: Spinal cord, Uninjured, P6981_1044


title: Spinal cord, Uninjured, P6981_1041


title: Spinal cord, Uninjured, P6981_1057


title: CUTNRUN_Zfp574_aV5_parental_ctl_cell_line_rep1

value: V5-tag antibody (Thermo Fisher, R960-25)


title: Spinal cord, Uninjured, P6981_1039


title: Spinal cord, Uninjured, P6981_1040


title: Pt-16 [Genotyping GoT-ChA amplicon]

city: New York

first: Franco

last: Izzo

state: NY

address: 1470 Madison Avenue

department: Oncological Sciences

institute: Icahn School of Medicine at Mount Sinai

description: Amplicons obtained from the GoT-ChA library

data_processing:
FASTQ files were processed through Gotcha (v1.0.0) and GotchaMito (v1.0.0)
Assembly: hg38
Supplementary files format and content: Processed data included in the file MetadataPatients.csv, available on series record.

source_name: Peripheral blood mononuclear cells (PBMC)

growth_protocol: Human CA46 (ATCC, #CRL-1648), HEL (ATCC, #TIB-180), SET-2 (DSMZ, #ACC 608) and CCRF-CEM (ATCC, #CRM-CCL-119) cell lines were maintained according to standard procedures in RPMI-1640 (Thermo Fisher Scientific, #11-875-119) with 10% (or 20% for CA46 and SET-2 cells) FBS (Thermo Fisher Scientific, #10-437-028) at 37˚C with 5% CO2. Cell lines in culture were screened biweekly for mycoplasma contamination using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza, #LT07-703).

extract_protocol:
Patient samples were FACS-sorted using CD34+ DAPI- gating. Downstream processing using the 10X Chromium Solution platform for single cell ATAC sequencing, with the modifications of the GoT-ChA protocol.
Nuclei were subsequently processed according to the Chromium Next GEM Single Cell ATAC Solution user guide (version CG000209 Rev F, 10x Genomics) with the following modifications: 1- During the GEM Generation and Barcoding reaction (Step 2.1), 1 μL of 22.5 μM GoT-ChA primer mix was added to the barcoding reaction mixture. The primers used are GoT-ChA_R1N (locus-specific primer with a Read 1N handle sequence: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG - [22bp locus specific]) and GoT-ChA_IS2 (locus- specific primer with an IS2 handle sequence: AGCAAGTGAGAAGCATCGTGTC - [22bp locus specific]). These primers allow for exponential amplification of the GoT-ChA fragments, relative to the linear amplification of ATAC fragments. 2- During the Post GEM Incubation Cleanup (Step 3.2), 45.5 μL of Elution Solution I is used to elute material from SPRIselect beads. 5 μL is used for GoT-ChA library construction, and the remaining 40 μL are used for ATAC library construction as indicated in the standard protocol. 3 - To generate the GoT-ChA library, two additional PCRs were performed on the 5 μL set aside during Step 3.2. The first PCR aims to amplify genotyping fragments prior to sample indexing and uses P5 (binds the P5 Illumina sequencing handle: AATGATACGGCGACCACCGAGATCTACAC) and GoT- ChA_nested (a nested, biotinylated, locus specific primer with a TruSeq Small RNA Read 2 handle: /5BiosG/CCTTGGCACCCGAGAATTCCA-[22bp locus specific]) primers with the following thermocycler program: 95 °C for 3 min; 15 cycles of 95 °C for 20 s, 65°C for 30 s and 72°C for 20 s; followed by 72°C for 5 min and ending with hold at 4°C. After a 1.2X SPRIselect clean up, biotinylated PCR product is bound and isolated using Dynabeads M-280 Streptavidin magnetic beads (Thermo Fisher Scientific, #11206D). Briefly, beads are washed three times with 1X sodium chloride-sodium phosphate-EDTA buffer (SSPE, VWR, #VWRV0810- 4L), added to the purified PCR product, and incubated at room temperature for 15 minutes. The beads are then washed twice with 1X SSPE buffer and once with 10 mM Tris-HCl (pH 8.0) before resuspending in water. The bead-bound fragments are then amplified and sample indexed using P5 and RPI-X (binds the TruSeq Small RNA Read 2 handle and adds a sample index and P7 Illumina sequencing handle: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGAC TGGAGTTCCTTGGCACCCGAGAATTCCA, “X” denotes user-defined sample index) primers with the following thermocycler program: 95 °C for 3 min; 6- 10 cycles of 95 °C for 20 s, 65°C for 30 s and 72°C for 20 s; followed by 72°C for 5 min and ending with hold at 4°C
ATAC libraries were sequenced to a depth of 25,000 read pairs per nucleus and GoT-ChA libraries were sequenced to 5,000 read pairs per nucleus

value: Untreated


title: Pt-19 [Genotyping GoT-ChA amplicon]

value: Peripheral blood mononuclear cells (PBMC)


title: Pt-17 [Genotyping GoT-ChA amplicon]


title: Pt18 [Genotyping GoT-ChA amplicon]

value: Ruxolitinib


title: Pt-15 [Genotyping GoT-ChA amplicon]


title: CUTNRUN_Zfp574_aV5_AID_cell_line_rep1

description:
CUTNRUN_Zfp574_aV5_AID_cell_line_merged.bw
CUTNRUN_peaks_Zfp574_aV5_AID_cell_line_merged.narrowPeak


title: Spinal cord, Uninjured, P6981_1038

value: EGFP+; tdTomato-


title: Pt-14 [Genotyping GoT-ChA amplicon]


title: Spinal cord, Uninjured, P6981_1042


title: Spinal cord, Uninjured, P6981_1037


title: Spinal cord, Uninjured, P6981_1036


title: Spinal cord, Uninjured, P6981_1035


title: CUTNRUN_Zfp574_aV5_AID_cell_line_rep2


title: Pt-12 [Genotyping GoT-ChA amplicon]


title: mouse_repressor_sorted_rep1

description:
mouse_repressor_sorted_rep1_counts_same_strand.txt
mouse_repressor_sorted_rep1_counts_rev_strand.txt
mouse_repressor_screen_sorted_vs_unsorted.txt

data_processing:
First, iPCR reads from sorted and background (non-selected) samples were trimmed using Trim galore (v0.6.0) with default parameters to remove Illumina adapters. Then, trimmed reads were aligned to the mm10 version of the mouse genome (mouse assays) or to the hg38 version of the human genome (human assays) using bowtie216 with default parameters (for paired-end sequenced samples, only first mate reads were considered), before removal of duplicated and low mapping quality reads (mapq<=30) using samtools (v1.9)17. Mapped insertions were assigned to the closest downstream exon junction – with a maximum distance of 200kb – based on GENCODE annotations of the mouse genome (vM25, mouse assays) or of the human genome (v43, human assays). Finally, insertion counts were aggregated per gene. Of note, only exons from protein-coding transcripts were considered, except for the first exon of each transcript, which might not contain splicing acceptor sites. Consequently, intronless genes – for which none of the isoforms contain a spliced intron – were not considered.
For each species, background replicates showed reproducible gene counts and therefore were merged, and genes with at least one insertion were considered as putatively tagged. Finally, genes showing significantly more insertions in sorted samples compared to merged background samples were identified using one-tailed fisher's exact test (alternative= "greater") on merged biological replicates. Of note, only genes with at least 3 unique insertions in sorted samples were considered. Obtained p-values were corrected for multiple testing using the FDR method and genes showing an FDR<0.001 and a log2 Odd Ratio≥1 were classified as hits.
Assembly: mm10 (mouse) and hg38 (human)
Supplementary files format and content: counts_same_strand files contains the genomic coordinates of all the insertion that were found in the sample, and the id of the assigned gene. In addition, the distance between the insertion and the exon that was used for assignment, as well as its position (number) inside the gene, are reported.
Supplementary files format and content: counts_rev_strand is similar to the previous (counts_same_strand) file, but report the assignment of reversed insetions. Used to compute strand bias.
Supplementary files format and content: The sorted_vs_unsorted.txt files contain, for each gene, the unique insertions enrichment in sorted vs unsorted samples (log2OR) with the associated FDR (padj). In this study, all genes with a FDR<0.001 were considered as hits (see "hit" column).
Library strategy: ORFtag

growth_protocol: All mouse experiments presented here were carried out in diploid mouse embryonic stem cells (mESCs) that were derived from originally haploid HMSc2 termed AN3-128. The mESCs were cultivated without feeders in high-glucose-DMEM (Sigma-Aldrich) supplemented with 13.5% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 1x Penicillin-Streptomycin (Sigma-Aldrich), 1x MEM non-essential amino acid solution (Gibco), 1mM sodium pyruvate (Sigma-Aldrich), 50 mM β-mercaptoethanol (Merck) and in-house produced recombinant LIF. Human ORFtag experiments were carried out in RKO (activator screen, ATCC, CRL-2577) or HEK293 (repressor screen, ATCC, CRL-1573) and the cells were cultivated in high-glucose-DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 1x Penicillin-Streptomycin (Sigma-Aldrich). Virus packaging cell lines, Lenti-X 293T (Takara), PlatinumE (Cell Biolabs) and PlatinumA (Cell Biolabs), were grown according to the manufacturer’s instructions. All cell lines were cultured at 37°C and 5% CO2 and regularly tested for mycoplasma contamination.

extract_protocol:
Genomic locations of ORFtag integrations were mapped using modified inverse-PCR followed by next generation sequencing (iPCR-NGS) protocol8. Genomic DNA was prepared by lysing cell pellets in lysis buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg/ml proteinase K) at 55°C overnight. Following a 2-hour RNase A treatment (Qiagen, 100 mg/ml, 1:1,000 dilution) at 37°C, two extractions using phenol:chloroform:isoamyl alcohol and one extraction using chloroform:isoamyl alcohol were carried out.
The samples then underwent two separate digestion reactions (with up to 4 µg of genomic DNA) using NlaIII and MseI enzymes (NEB) at 37°C overnight, followed by purification using a Monarch PCR&DNA Cleanup Kit (NEB). Ring-ligation was carried out using T4 DNA ligase (NEB) at 16 °C overnight, followed by heat-inactivation (65°C, 15 min) and linearization using SbfI-HF (NEB) at 37°C for 2 h. The digests were then purified using a Monarch PCR&DNA Cleanup Kit (NEB) and amplified using firstly a nested PCR reaction with KAPA HiFi HotStart ReadyMix (Roche), and a specific primer pair (TGCAGGACCGGACGTGACTGGAGTTC*A, TGCAGGACGATGAGCAGAGCCAGAACC*A) for 16 cycles. After cleanup with AMPure XP Reagent (Beckman Coulter, 1:1 ratio beads:PCR), iPCR amplification was carried out with KAPA HiFi HotStart ReadyMix (Roche), and a specific primer pair (AATGATACGGCGACCACCGAGATCTACACGAGCCAGAACCAGAAGGAACTTGA*C, CAAGCAGAAGACGGCATACGAGAT [custom-barcode] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) for 18 cycles. Afterwards, amplified libraries were size selected for a range of 400-800 bp using SPRIselect beads (Beckman Coulter). NGS was performed on an Illumina NextSeq550 or llumina HiSeq 2500 sequencer according to the manufacturers’ protocols with custom first-read primer (1:1 mix of GAGTGATTGACTACCCGTCAGCGGGGGTCTTTCA and TGAGTGATTGACTACCCACGACGGGGGTCTTTCA).

treatment_protocol: The ORFtag viral constructs were derived from the ecotropic Retro-EGT construct8 that includes the sequence features necessary for the inverse-PCR protocol (see below). Furthermore, the construct contains constitutively active PGK promoter that drives the expression of a NeoR resistance gene separated from a tag by the IRES sequence. The tag contained either TetR with an N-terminally located nuclear localization signal (Activator and Repressor screens; Addgene, this study) or LambdaN domain (PTGR screen; Addgene, this study). Additionally, the tag contained 2x GGGS-linker followed by the BC2-tag and 3xFLAG-tag. Finally, the ORFtag construct contains a consensus splice donor motif followed by a part of the Hprt intron (chrX:53020400-53020556 +, mm10). In order to tag genes in all three possible coding frames, three constructs were used that contain either 0, 1 or 2 additional nucleotides upstream of the consensus splice motif. Retroviral constructs carrying ORFtag cassette were packed in PlatinumE cell lines (for the screens performed in mESCs) or PlatinumA cell lines (for the screen performed in HEK293 cells) using polyethylenimine (PEI) reagent as described previously8. Reporter cell lines (100-150 million cells) were transduced with packaged retrovirus in the presence of 6 µg/ml polybrene (Sigma) and at low transduction efficiency (< 20%) to ensure only one virus per cells. Cells were harvested 24 hours later and plated in medium containing 0.1 mg/ml G418 (Gibco) for selection of transduced cells. Selection was continued until all cells on the control plate died (4-5 days), after which 40 million cells were processed as non-selected background for mapping of genomic integrations (see below). The remaining cells were sorted for GFP-positive (mouse and human Activator screens) or GFP-negative (mouse and human Repressor screens) populations using BD FACSAria III or IIu cell sorters (BD Biosciences) and processed for mapping of genomic integrations (see below). For the PTGR screen a five-sort strategy was applied to enrich cells that show a tethering dependent repression of reporter gene expression. Cells with a GFP expression equal to the lowest 10 percent of GFP expression observed after selection were sorted using BD FACSAria III and expanded thereafter. Additionally, non-sorted cells were maintained for gating of the consecutive sorts. Two additional sorts for cells with GFP expression similar to the lowest 10% of GFP signal observed in the non-sorted cells were performed and again expanded in-between the sorts. A fourth sort was performed for cells with a GFP expression equal to the lowest 5% of GFP signal observed in the non-sorted cells. After expansion, the cells were treated with 500nM 4-Hydroxytamoxifen (Sigma) to induce Cre-mediated recombination and to flox the boxB sites of the reporter construct and hence to revert the tethering. Thereafter a final sort was performed to select a cell population with a GFP expression equal to the highest 70% of GFP expressing cells. Transduction efficiency was measured by plating 10,000 cells on a 15-cm dish and selecting with G418 (Gibco). A control plate with 1,000 cells was also plated without selection. After 10 days, colonies were counted and transduction efficiency was calculated as the number of colonies on the selected plate divided by the total number of cells plated (10 times the number of colonies on the control plate).

value: wild-type cells with pEF1-BFP-7xTetO-pPGK-Puro-IRES-GFP reporter integrated into the expression-stable locus on Chr15, clonal cell line

value: sorted


title: Pt-11 [Genotyping GoT-ChA amplicon]


title: Spinal cord, Uninjured, P6981_1033


title: mouse_activating_unsorted_rep2

description:
mouse_activating_unsorted_rep2_counts_same_strand.txt
mouse_activating_unsorted_rep2_counts_rev_strand.txt
mouse_activating_screen_sorted_vs_unsorted.txt

value: wild-type cells with 7xTetO-pMYLPF-Puro-IRES-GFP reporter integrated into the expression-stable locus on Chr15, clonal cell line

value: unsorted


title: Spinal cord, Uninjured, P6981_1034


title: Pt-09 [Genotyping GoT-ChA amplicon]


title: Pt-13 [Genotyping GoT-ChA amplicon]


title: Spinal cord, Uninjured, P6981_1031


title: Multiplexing_adapted [Genotyping GoT-ChA amplicon]

description: Cell line mixture experiment to determine multiplexing capacity in adapted setting

source_name: Cell mixing experiment containing HEL, SET-2, CA46 and OCI-AML3 cell lines targeting JAK2-V617F, TP53-M133, NRAS-Q61and TP53-R248 using adapted GoT-ChA protocol for multiplexing

value: Cell line mixture 1:1:1:1 (HEL, SET-2, CA46 and OCI-AML3)


title: Pt-10 [Genotyping GoT-ChA amplicon]


title: mouse_activating_unsorted_rep1

description:
mouse_activating_unsorted_rep1_counts_same_strand.txt
mouse_activating_unsorted_rep1_counts_rev_strand.txt
mouse_activating_screen_sorted_vs_unsorted.txt


title: mouse_PTGR_sorted_rep2

description:
mouse_PTGR_sorted_rep2_counts_same_strand.txt
mouse_PTGR_sorted_rep2_counts_rev_strand.txt
mouse_PTGR_screen_sorted_vs_unsorted.txt

value: wild-type cells with Puro-IRES-GFP-10xBoxB reporter randomly integrated, clonal cell line


title: Multiplexing_original_protocol [Genotyping GoT-ChA amplicon]

description: Cell line mixture experiment to determine multiplexing capacity

source_name: Cell mixing experiment containing HEL, SET-2, CA46 and OCI-AML3 cell lines targeting JAK2-V617F, TP53-M133, NRAS-Q61and TP53-R248


title: mouse_repressor_sorted_rep2

description:
mouse_repressor_sorted_rep2_counts_same_strand.txt
mouse_repressor_sorted_rep2_counts_rev_strand.txt
mouse_repressor_screen_sorted_vs_unsorted.txt


title: Spinal cord, Uninjured, P6981_1028

value: Spinal cord

value: Perivascular cells


title: mouse_activating_sorted_rep2

description:
mouse_activating_sorted_rep2_counts_same_strand.txt
mouse_activating_sorted_rep2_counts_rev_strand.txt
mouse_activating_screen_sorted_vs_unsorted.txt


title: Spinal cord, Uninjured, P6981_1032

value: female


title: Spinal cord, Uninjured, P6981_1024


title: Pt-08 [Genotyping GoT-ChA amplicon]


title: mouse_activating_sorted_rep1

description:
mouse_activating_sorted_rep1_counts_same_strand.txt
mouse_activating_sorted_rep1_counts_rev_strand.txt
mouse_activating_screen_sorted_vs_unsorted.txt

value: mESC


title: mouse_PTGR_unsorted_rep2

description:
mouse_PTGR_unsorted_rep2_counts_same_strand.txt
mouse_PTGR_unsorted_rep2_counts_rev_strand.txt
mouse_PTGR_screen_sorted_vs_unsorted.txt


title: mouse_repressor_unsorted_rep2

description:
mouse_repressor_unsorted_rep2_counts_same_strand.txt
mouse_repressor_unsorted_rep2_counts_rev_strand.txt
mouse_repressor_screen_sorted_vs_unsorted.txt


title: mouse_repressor_unsorted_rep1

description:
mouse_repressor_unsorted_rep1_counts_same_strand.txt
mouse_repressor_unsorted_rep1_counts_rev_strand.txt
mouse_repressor_screen_sorted_vs_unsorted.txt


title: GN031_-iM0 hECT

city: NEW YORK

first: Gordana

last: Vunjak-Novakovic

department: Biomedical Engineering

institute: Columbia University

data_processing:
RTA (Illumina) was used for base calling and bcl2fastq2 (version 2.19) for converting BCL to fastq format, coupled with adaptor trimming
Pseudoalignment to a kallisto index created from transcriptomes (Ensembl v96, Human:GRCh38.p12) was performed using kallisto (0.44.0)
Estimated counts and tpm values for each gene are computed from tximport R package.
Assembly: GRCh38.p12
Supplementary files format and content: Tab separated values (tsv) files are plaintext files of the abundance estimates. Includes estimated counts, TPM, effective length for each transcript.
Supplementary files format and content: Javascript object notation (JSON) files contain the run information for each sample.
Supplementary files format and content: Tab delimited text file contains estimated counts for each gene for all samples, computed from tximport R package.
Supplementary files format and content: Tab delimited text file contains tpm values for each gene for all samples, computed from tximport R package.

source_name: human engineered cardiac tissue

growth_protocol: All cell types were differentiated from human iPSCs. For -iM0 human engineered cardiac tissues (hECT), cardiomyocytes (iCM) and fibroblasts (iCF) were encapsulated in a fibrin hydrogel around flexible pillars. For +iM0 hECT, iCM, iCF, and macrophages (iM0) were combined and encapsulated in a fibrin hydrogel around flexible pillars. All groups were cultured in RPMI basal medium with B27 supplement, which was refreshed every two days. Tissues were cultured over a period of 28 days from initial tissue fabrication.

extract_protocol:
Tissues were snap frozen in liquid nitrogen and stored at -80 ºC until use. To isolate RNA, snap-frozen tissues were added to 300 µL of RNA lysis buffer with a stainless-steel bead (BioSpec, cat. no. 11079123) and homogenized with a Mini-Beadbeater (Biospec, cat. no. C321001) for two cycles of10 second intervals, with storage on ice between cycles. A Qiagen RNeasy Micro Kit (Qiagen, cat. no. 74004) was then used according to the manufacturer's instructions. Total RNA for each sample was quantifiedusing an Agilent 2100 Bioanalyzer with an RNA Pico Chip at Columbia’s Molecular Pathology core. RNA samples with an RNA integrity number (RIN) > 8 were considered acceptable for further analysis.
A library was prepared through Columbia’s Genome Center using a Nextera XT DNA Library Preparation kit (Illumina TruSeq Chemistry) with 100-150pg starting material, following the manufacturer's instructions.

treatment_protocol: Following tissue fabrication, after 7 days of culture, tissues were subjected to a 2-week frequency-ramped electromechanical stimulation regimen, then continuously paced at 2 Hz until the end of the 28 day culture period.

value: human engineered cardiac tissue

value: WTC11 iPSC

value: iPSC-derived cardiomyocytes and cardiac fibroblasts

value: WT

value: cardiomyocyte and cardiac fibroblast differentiation from iPSC, suspension in fibrin hydrogel, and electromechanical stimulation of tissue in culture.


title: GN033_-iM0 hECT


title: Spinal cord, Uninjured, P6981_1021

source_name: Spinal cord


title: GN036_+iM0 hECT

value: iPSC-derived cardiomyocytes, cardiac fibroblasts, and macrophages

value: cardiomyocyte, cardiac fibroblast, and macrophage differentiation from iPSC, suspension in fibrin hydrogel, and electromechanical stimulation of tissue in culture.


title: Spinal cord, Uninjured, P6981_1027

value: EGFP+; tdTomato-


title: MKs, Adult peripheral blood sample 3, rep 2

city: Philadelphia

first: Christopher

last: Thom

state: PA

address: 3501 Civic Center Blvd

institute: Children's Hospital of Philadelphia

hyb_protocol: Affymetrix protocol

scan_protocol: Affymetrix protocol

data_processing:
R/Bioconductor (oligo, limma, affycoretools) were used in the initial processing.
File containing log2-fold changes in expression (PB_vs_CB.results.txt).

label: biotin

source_name: Adult peripheral blood-derived megakaryocytes

label_protocol: Affymetrix protocol

extract_protocol: Total RNA was isolated using commercially available kits.

value: Adult peripheral blood MKs


title: GN035_+iM0 hECT


title: resistant, 5 [RNA-seq]

city: Little Rock

first: Stephanie

middle: D

last: Byrum

state: Arkansas

address: 501 Jack Stephens Dr, Jackson T. Stephens Bldg 4th floor

institute: University of Arkansas for Medical Sciences

description: pdx-human/mouse

data_processing:
RNA-seq reads were quality-checked, trimmed, and aligned to the hg38 reference genome (accession: GCA_000001405.15) and our custom hg38 genomes using the Nextflow RNAseq pipeline, nf-core/rnaseq (version 3.4) available at DOI 10.5281/zenodo.1400710.
Assembly: GCA_000001405.15 (hg38)
Supplementary files format and content: Comma-separated text file includes counts.

source_name: NRASQ61L melanoma tumor

extract_protocol:
Genomic DNA (gDNA) and total RNA were extracted from snap frozen patient tumor and patient-matched normal tissue, and frozen PDX tumors preserved in RNALater using the QIAGEN AllPrep DNA/RNA Mini Kit and the Ambion mirVana miRNA Isolation Kit.
RNA libraries were constructed using the NuGen Universal Plus mRNA-Seq Kit. Briefly, after fragmentation of total RNA and double-stranded cDNA generation using a mixture of random and oligo(dT) primers, the RNA libraries were constructed by end repairing, adapter ligation, strand selection, and PCR amplification. After RNA library construction, libraries were quantified for equal molar pooling and paired-end sequenced with a read length of 2×50 bp on the Illumina NovaSeq 6000 S2 platform.

value: NRASQ61L melanoma tumor

value: trametinib


title: Spinal cord, Uninjured, P6981_1026

data_processing:
Clustering was done by 'cBot' and samples were sequenced on Illumina HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 1x51 setup using 'HiSeq SBS Kit v4' chemistry. The Bcl to FastQ conversion was performed using bcl2fastq from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+. Mus musculus, GRCm38 was used as reference genome.   We used a single-cell RNA sequencing snakemake workflow (“lts_workflows_sm_scrnaseq”) for alignment, quantification and quality control using resources as follows: The reads were aligned to the mouse (mm10) genome using STAR 2.5.3a (Dobin et al., 2013) and transcripts quantified in terms of Reads Per Kilobase of transcript per Million mapped reads (RPKM) using rpkmforgenes v1.0.1  (Ramsköld et al., 2009) and RSEM to estimate gene and isoform expression levels v1.2.28  (Li and Dewey, 2011). RSeQC V2.6.4  (Wang et al., 2012) Multiqc v0.9.1 were used for quality control. To filter out low quality libraries the following filtering criteria for quality control were applied: star_Uniquely mapped: < mean - 2 sd, gene_detection : > mean - 2 sd & < mean + 2 sd, count_gene : < mean - 2 sd, gene_correlation_max : < mean - 2 sd. Cells that failed for 2 of the criteria were filtered out (25 cells, 2.89% of 864, the total number of cells).
Assembly: mm10
Supplementary files format and content: rpkmforgenes_counts.tab
Supplementary files format and content: 170706_sampleinfo_scs.csv

extract_protocol:
Preparation of single-cell cell suspension and myelin removal was adapted from previously published protocols (Lovatt et al., 2007; Orre et al., 2014). In summary, the procedure was: 5 days after spinal cord crush injury, animals were euthanized by intraperitoneal injection of sodium pentobarbital (200mg/Kg, 100ul i.p., APL) and transcardially perfused with cold Hank's Balanced Salt Solution without calcium or magnesium (HBSS) (Invitrogen). The vertebral column was immediately removed to cold HBSS, the spinal cord was carefully dissected out of the vertebrae and the crush injury segment was separated from the uninjured spinal cord and treated separately hereafter. After removal of the outer meningeal layers, the tissue was dissociated mechanically with a razor blade on a glass petri dish and immediately transferred to cold HBSS. Followed by enzymatic digestion with papain at a final concentration of 8 U/mL with 80 Kunitz units/mL DNase I (Sigma-Aldrich, D4263) in Ca2+/Mg2+-free piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)/cysteine -based buffer, pH 7.4, for 40min at 37°C, 350rpm on a ThermoMixer® (Eppendorf). The tissue was carefully triturated and incubated for additional 5min. The cell suspension was passed through a 70μmCell Strainer (Corning), washed with Minimum Essential Media (MEM) with 1% bovine serum albumin (BSA) and spun down at 200g for 5min at 4°C. The cells were resuspended in MEM and centrifuged over a 90% Percoll gradient (GE Healthcare, 17-0891-01) at 250g for 15min at 4°C. Cells in the lipid layer and below were diluted 5 times in MEM with 1% BSA and spun in a 15mL tube at 250g for 10min at 4°C. All supernatant including the lipid layer were carefully removed and the pellet resuspended in cold MACS Buffer and magnetic Myelin removal beads (Miltenyi biotech, 130-096-433) and incubated for 15min at 4°C. The cells were washed and run over MACS MS columns (Miltenyi biotech, 130-042-201) on a magnetic stand according to the manufacturer’s instructions. The flow through cells were collected, spun down and resuspended in FACS buffer. The single cell suspensions derived from normal spinal cord and injury segments were subject to FACS (fluorescence activated cell sorting) into GLAST-CreER-tomato+/Pdgfrb-eGFP+ double positive and Pdgfrb-eGFP+ single cells, into 96-well plates with Cell lysis buffer (Clonetech, 635013) and frozen to -80°C. Cell sorting was performed on a FACSAria III Cell Sorter system (BD Biosciences). Singlet discrimination was performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H), dead cells were excluded by SYTOX Blue Dead Cell Stain (ThermoFisher Scientific, S34857).
Single cells were processed to cDNA libraries according to the Smart-Seq2 protocol  (Picelli et al. 2014). Tn5 tagmentation was performed using the Nextera XT DNA library Preparation Kit (Illumina, FC-131-1002) using dual indexes (i7 and i5).


title: GN034_+iM0 hECT


title: Spinal cord, Uninjured, P6981_1025


title: MKs, Adult peripheral blood sample 4, rep 1


title: resistant, 2 [RNA-seq]

description: pdx-human/mouse


title: Spinal cord, Uninjured, P6981_1030


title: Spinal cord, Uninjured, P6981_1019


title: Spinal cord, Uninjured, P6981_1022


title: resistant, 1 [RNA-seq]

description: pdx-human/mouse


title: MKs, Adult peripheral blood sample 4, rep 2


title: GN032_-iM0 hECT


title: MKs, Adult peripheral blood sample 1, rep 1


title: Spinal cord, Uninjured, P6981_1023


title: Spinal cord, Uninjured, P6981_1029


title: Spinal cord, Uninjured, P6981_1018


title: MKs, Adult peripheral blood sample 3, rep 1


title: resistant, 4 [RNA-seq]

description: pdx-human/mouse


title: MKs, Cord blood sample 5, rep 1

source_name: Umbilical cord blood-derived megakaryocytes

value: Cord blood MKs


title: Spinal cord, Uninjured, P6981_1017


title: Spinal cord, Uninjured, P6981_1020


title: resistant, 4

description: pdx-human/mouse

data_processing:
Fastq files were trimmed using trimmomatic and reads with Q>30 were kept. Reads were aligned to the hg38 reference genome (accession: GCA_000001405.15) using bowtie2. Somatic mutations were identified using Mutect222 and Strelka223 variant callers for V compared to normal, R1 vs. normal, R2 vs. normal, R4 vs. normal, and R5 vs. normal with hg38 as reference. The intersection of Mutect2 and Strelka2 variant calling algorithms was used to identify the variants within each tumor, which were used for further downstream analyses. Additionally, the intersection of variants between resistant tumors was identified to find a common set of variants in the resistance group. The variants were filtered to retain only passing ones. The resulting variants were used to build uniformly annotated, custom hg38 reference genomes using Bcftools (version 1.9)24. These custom hg38 genomes were used as references for gene and protein analyses. Additionally, the variant files were uniformly annotated and the resulting protein sequences of called variants were obtained using SnpEff (version 5.0e)25.
Assembly: GCA_000001405.15 (hg38)
Supplementary files format and content: vcf files
Library strategy: WES

extract_protocol:
Genomic DNA (gDNA) and total RNA were extracted from snap frozen patient tumor and patient-matched normal tissue, and frozen PDX tumors preserved in RNALater using the QIAGEN AllPrep DNA/RNA Mini Kit and the Ambion mirVana miRNA Isolation Kit.
gDNA libraries were constructed using the Roche NimbleGen SeqCap EZ Human Exome Library Kit v2.0, which captures 64 Mb of the human genome by 2.1 million oligonucleotide probes for coverage of more than 20,000 genes in the human genome (Roche NimbleGen, Inc., Madison, WI). Briefly, after fragmentation of gDNA, the libraries were constructed by end repairing and A-tailing the fragmented DNAs, ligation of adapters, and PCR amplification.


title: vehicle, 1 [RNA-seq]

description: pdx-human/mouse

value: vehicle


title: MKs, Cord blood sample 4, rep 3


title: MKs, Cord blood sample 4, rep 4


title: resistant, 5

description: pdx-human/mouse


title: Spinal cord, Uninjured, P6981_1014

value: Spinal cord


title: MKs, Cord blood sample 5, rep 2


title: MKs, Adult peripheral blood sample 2, rep 1


title: MKs, Adult peripheral blood sample 1, rep 2


title: normal

description:
pdx-human/mouse
This sample was used for variant calling in all other samples.

source_name: normal tissue

value: normal tissue


title: Spinal cord, Uninjured, P6981_1013

value: Perivascular cells

value: female


title: resistant, 1

description: pdx-human/mouse


title: Spinal cord, Uninjured, P6981_1011


title: Spinal cord, Uninjured, P6981_1016


title: vehicle,1

description: pdx-human/mouse


title: resistant, 2

description: pdx-human/mouse


title: Spinal cord, Uninjured, P6981_1009


title: Spinal cord, Uninjured, P6981_1010


title: MKs, Cord blood sample 4, rep 1


title: p16_high 2

city: Suwon

first: Soon Sang

last: Park

address: Worldcup-Ro 206

country: South Korea

institute: Ajou University School of Medicine

data_processing:
Raw quality bases in the raw reads were trimmed by Skewer ver 0.2.2
Raw quality bases in the raw reads were mapped to the reference genome by STAR ver 2.5 software.
To quantify the mapped reads on the reference genome into the gene expression values, Cuffquant in Cufflinks ver 2.2.1 was used.
Assembly: hg38
Supplementary files format and content: csv file include RPKM values for each sample.

growth_protocol: The fresh frozen tissue from Glioblastoma, IDH-wild type grade 4 patients is surgically removed at Ajou University Hospital, Korea.

extract_protocol:
The 30mg of fresh frozen tissue was homogenized using mortar and pestle. RNA was extracted using Nucleospin RNA (Macherey-Nagel). 1ug of RNA was used to make sequencing libraries.
RNA libraries were prepared using TruSeq Stranded mRNA Prep Set following manufacturer's protocol.

value: Brain

tag: condition


title: MKs, Cord blood sample 4, rep 2


title: Spinal cord, Uninjured, P6981_1015


title: p16_high 3


title: MKs, Cord blood sample 3, rep 2


title: p16_high 1


title: p16_low 4


title: Spinal cord, Uninjured, P6981_1012


title: p16_low 5


title: p16_low 3


title: Spinal cord, Uninjured, P6981_1007


title: p16_low 2


title: MKs, Cord blood sample 3, rep 4


title: MKs, Cord blood sample 2, rep 1


title: Spinal cord, Uninjured, P6981_1006


title: MKs, Cord blood sample 3, rep 3


title: MKs, Cord blood sample 3, rep 1


title: MKs, Cord blood sample 1, rep 2


title: MKs, Cord blood sample 1, rep 1


title: Spinal cord, Uninjured, P6981_1004


title: p16_low 1


title: cord blood-derived megakaryocytes, 96h DMSO 2

data_processing:
R/Bioconductor (oligo, limma, affycoretools) were used in the initial processing.
File containing log2-fold changes in expression (CA96h.results.txt).

value: Umbilical cord blood-derived megakaryocytes

value: DMSO for 96 h


title: Perilesional normal 3

value: normal


title: Perilesional normal 2


title: cord blood-derived megakaryocytes, 96h CA 3

value: Cortistatin A for 96 h


title: Spinal cord, Uninjured, P6981_1005


title: Spinal cord, Uninjured, P6981_1002


title: Spinal cord, Uninjured, P6981_1008


title: cord blood-derived megakaryocytes, 96h DMSO 3


title: Perilesional normal 1


title: Patient7, post Treatment, TCRseq

city: Berlin

first: Artur

last: Wilhelm

address: Virchowweg 11

department: Rheumatology and clinical immunology

institute: Charite Berlin

description: 10x Genomics

library_source: other

data_processing:
Demultiplexing, barcode processin, gene counting and clonotype calling was performed using the cellranger multi pipeline v.7.0.0.
Assembly: hg38
Supplementary files format and content: Tab seperated matrix, gene and barcode files
Library strategy: TCR-Seq

molecule: other

source_name: Blood

extract_protocol:
Peripheral blood mononuclear cells (PBMCs) were separated using SepMate™-50 (StemCell) and Ficoll-Paque gradient centrifugation following the manufacturer's protocol.
libraries were constructed using the Chromium Next GEM Single cell 5´Reagent Kits v2 (10 X Genomics)

value: Blood

value: T cells

value: post anti CD19 CART-cell therapy


title: RNASeq_activating_unsorted

data_processing:
First, ORFtag RNA-seq reads were separated depending on the frame of the cassette, using regular expression matching anchored at the start of reads’ sequence (frame 1: ^[NACGT]{6}CCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGCAG, frame 2: ^[NACGT]{6}CCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGGCAG, frame 3: ^[NACGT]{6}CCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGGCCAG), allowing for one mismatch. Then, the constant sequences were trimmed before mapping the remaining part of the reads – corresponding to the first spliced exon — to the mm10 version of the mouse genome.
Finally, reads were assigned to their overlapping genomic features (exons, introns, upstream of TSS (up to 200kb) and intergenic regions) in order to infer the phase of the transcript (see transcriptPhase column).
Assembly: mm10
Supplementary files format and content: RNASeq_activating_screen_reads_assignment_sorted.txt and *_unsorted.txt files contain, for each unique mappeg genomic coordinates (seqnames, start, end, strand) a unique ID (readID), the read counts, the type of overlapping genomic feature (exon, transcript (meaning intron), upstream of TSS (up to 200kb) and intergenic),  the exon_number of overlapping exon(s), the gene_id of overlapping gene(s), the expected phase (inferred from the ORFtag cassette corresponding to the transcript) and the transcriptPhase (inferred from the mapping position and the frame of overlapping exons). Of note, the readID is useful in the cases where an insertion is assigned to several exons or transcripts. Only the RNAseq reads for which expectedPhase =  transcriptPhase are expected to produce in-frame products.

growth_protocol: All experiments presented here were carried out in diploid mouse embryonic stem cells (mESCs) that were derived from originally haploid HMSc2 termed AN3-1213. The mESCs were cultivated without feeders in high-glucose-DMEM (Sigma-Aldrich) supplemented with 13.5% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 1x Penicillin-Streptomycin (Sigma-Aldrich), 1x MEM non-essential amino acid solution (Gibco), 1mM sodium pyruvate (Sigma-Aldrich), 50 mM β-mercaptoethanol (Merck) and in-house produced recombinant LIF. Virus packaging cell lines, Lenti-X 293T (Takara) and PlatinumE (Cell Biolabs), were grown according to the manufacturer’s instructions. All cell lines were cultured at 37°C and 5% CO2 and regularly tested for mycoplasma contamination.

extract_protocol:
Subsequently, mRNA was isolated using Oligo-dT25 beads (Invitrogen, 61005), followed by TurboDNase I treatment (Invitrogen, AM2238) at 37 °C for 30 minutes. The purified mRNA was cleaned with AMPure XP beads (Beckman Coulter, A63882) at a ratio of 1:1.8 (RNA:beads), followed by reverse transcription using SuperScript III (Invitrogen, 18080093) with a random hexamer primer at the final concentration 0.2 µM (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN) and the following conditions: 25  °C for 5 min, 50 °C for 1 h, 70 °C for 15 min. Afterwards, cDNA was treated with RNaseA (Thermo Fisher, EN0531) at 37 °C for 1 hour, followed by clean-up using AMPure XP beads at a ratio of 1:1.4.
Library amplification was performed using a semi-nested PCR approach. Initially, 5 µl of cDNA was amplified in ten PCR reactions using KAPA HiFi HotStart ReadyMix (Roche) and the following primers (fw: TATGTGGCCTGGAGAAACAGCTA & rev: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) under the following conditions: 98 °C for 45 s; 12 cycles of 98 °C for 15 s, 65 °C for 30 s,  72 °C for 30 s; followed by 72 °C for 60 s. The first amplification was followed by clean-up using AMPure XP beads at a ratio of 1:1.4. Subsequently, a second PCR in ten reactions was performed using KAPA HiFi HotStart ReadyMix and the following primers (fw: CACGACGCTCTTCCGATCTNNNNNNCCACGACGGAGACTACAAGG & rev: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) under the following conditions: 98 °C for 45 s; 5 cycles of 98 °C for 15 s, 65 °C for 30 s, 72 °C for 30 s; followed by 72 °C for 60 s. This was followed by a clean-up using AMPure XP beads at a ratio of 1:1.4. The final library amplification was carried out using the KAPA HiFi HotStart Real-Time Library Amp Kit (Roche) with Illumina Truseq Small RNA library amplification kit primers (8 cycles of amplification). Finally, the amplified libraries were size selected for a range of 200-800 bp using SPRIselect beads (Beckman Coulter).

treatment_protocol: Expanded GFP-positive cells and background cells (40 million each) from Activator ORFtag screen were collected, and total RNA extraction was carried out using the RNeasy Maxi kit (Qiagen, 75162) with β-mercaptoethanol supplemented RLT buffer.

value: mESC

value: wild-type cells with 7xTetO-pMYLPF-Puro-IRES-GFP reporter integrated into the expression-stable locus on Chr15, clonal cell line

value: unsorted


title: Spinal cord, Uninjured, P6981_1003

value: EGFP+; tdTomato-


title: cord blood-derived megakaryocytes, 96h CA 1


title: cord blood-derived megakaryocytes, 96h CA 2


title: RNASeq_activating_sorted

value: sorted


title: Patient7, pre Treatment, TCRseq

description: 10x Genomics

value: pre anti CD19 CART-cell therapy


title: cord blood-derived megakaryocytes, 8 h CA treated, biological rep 2

data_processing: R/Bioconductor (oligo, limma, affycoretools) were used in the initial processing.

label: biotion

value: 8h CA


title: cord blood-derived megakaryocytes, 96h DMSO 1


title: cord blood-derived megakaryocytes, 8 h CA treated, biological rep 3